PREFACE
This work has been done in collaboration between University Toulouse III – Paul Sabatier (UPS), France (Laboratory of coordination chemistry, UPR 8241 (LCC)) and Taras Shevchenko National university of Kyiv (UNTS), Ukraine (Faculty of chemistry) between 2014 and 2018. Financial support from the French government, Ukrainian government and University Paul Sabatier are gratefully acknowledged.
I am very much thankful to my supervisor Dr. Pascal George Lacroix for encouraging and fruitful scientific conversations, for his openness and readiness to help both in scientific and life questions. I also appreciate he made possible my research visits to Mexico thus opening so many opportunities to know new people, culture and establish new scientific contacts. I would like also to say thank you to Dr. Lacroix for the time he spent to teach me a brand new field of chemistry for me – DFT computations.
Many thanks to my research advisor Professor Zoia Voitenko who is one of founders of the French-Ukrainian scientific collaboration. I am thankful to her for her strong believe in me, for always being sincere and thoughtful about my needs as a researcher and a person. Never being detached from the problems she is a nice support for me.
My sincere thanks to Professor Isabelle Malfant, who made possible my work in LCC in the group under her guidance. Thank you for your critical point of view, patience and objectivity. Truly, it was a big pleasure to work in your team. I would also like to express my thanks to Dr. Isabelle Sasaki, whose pieces of advice were always helpful in the synthetic part of my work. And I also must point out a big help that Marine Tassé kindly provided every time there was a technical question in the laboratory work. My thanks to Mathilde Bocé, Hasan Mohammed and Max Roose for creating a friendly work atmosphere in the laboratory.
A big part of the work has been done with the facilities and opportunities provided by researchers from Mexico. I appreciate an invaluable help of Professor
José Norberto Farfán García at UNAM and I am very thankful to him for making possible my comfort and fruitful stay in Mexico, for providing nice conditions for work and collaboration with researchers of his group and for interesting scientific discussions. I would like also to say thank you to Dr. Rosa Luisa Santillan Baca, and Professor Farfan’s big family for opening the possibilities to discover the culture of Mexico and for always being responsive and helpful.
I would like also to express many thanks to Professor Blas Flores Pérez, who has kindly provided the facilities for the research work in UNAM. I am very much thankful to PhD Alejandro Enriquez-Cabrera for his readiness to help in any question regarding research work or everyday life and for providing a valuable help in the questions of synthetic chemistry. I am also very much obliged to him and his big family for nice treatment and hosting me during my stay in Mexico. Many thanks to Margarita Romero-Ávila for her invaluable help and guidance in many complex problems in synthetic organic chemistry, for her support and sensitiveness. I would like to express my gratitude to Andrés Felipe Leon-Rojas, a student of Professor Farfan’s research group, for nice collaboration and help in synthetic part of the work.
I gratefully acknowledge Dr. Gabriel Ramos-Ortiz (CIO, Leon, Mexico) for his kind attention and solicitude and for providing conditions and facilities to conduct the optical measurements. I am thankful to Rodrigo Misael Barba-Barba and PhD Jayaramakrishnan Velusamy for substantial help in measuring the two-photon absorption parameters of the complexes.
I would like also to express my thanks to my parents Olexandr and Natalia, my relatives and friends for their love and support. Thank you for understanding and empathy!
My dear Olena, thank you for your unceasing concern for me, for your believe and for being my infinite support!
Valerii Bukhanko August, 2018
TABLE OF CONTENT
ABBREVIATIONS ... 6
INTRODUCTION ... 9
CHAPTER 1. LITRERATURE OVERVIEW ... 11
1.1 Nitric oxide: molecular structure and general chemical characteristic. ... 11
1.2 Nitric oxide biological role ... 14
1.3 NO generation from organic compounds ... 19
1.3.1 Natural pathway of nitric oxide production ... 19
1.3.2 Classical donors of nitric oxide based on organic compounds. ... 20
1.3.3 Nitrobenzenes as examples of photoactive NO-donors ... 26
1.4 NO-donors based on coordination compounds ... 31
1.4.1 General characteristics of nitrosyl complexes. ... 31
1.4.2 Study of metal-nitrosyl complexes by means of IR spectroscopy ... 36
1.4.3. Ruthenium-nitrosyl complexes as NO donors ... 38
1.4.4 Mechanism of nitric oxide release from ruthenium-nitrosyl complexes: theoretical studies ... 50
1.5 Two-photon absorption as a method of photosensitization of compounds within the therapeutic window ... 54
CHAPTER 2. THE REPLACEMENT OF CHLORO-LIGANDS WITH 2,2'-BIPYRIDINE IN {Ru-NO} COMPLEXES AND ITS EFFECT ON TPA OF COMPOUNDS ... 59
2.1 Comparison of electronic spectra of compounds bpy1, cis1 and trans1. ... 62
2.2 Study of the release of nitric oxide from the complex bpy1. ... 65
2.3 Determination of the oxidation state of ruthenium in photoproduct through the analysis of its electronic spectrum. ... 67
2.4 Investigation of two-photon absorption of cis2, trans2 and bpy2 compounds. ... 69
CHAPTER 3. PYRIDOISOINDOLE AND ISOMERIC HETEROCYCLES AS DONATING FRAGMENTS IN {Ru-NO} COMPLEXES: THEORETICAL INVESTIGATION. ... 75
3.1. Pyridoisoindoles and isomeric heterocycles as potentially more effective fluorene analogues ... 77
3.2. Quantum-chemical calculations of nonlinear optical parameters of
complexes. ... 80 3.3 Push-pull effects in studied complexes ... 82 3.4 Quadratic (β) against cubic (σTPA) properties of complexes. ... 86 CHAPTER 4. ENHANCEMENT OF THE EFFICIENCY OF RUTHENIUM-NITROSYL COMPLEXES IN TPA ... 90
4.1. Influence of the efficiency of conjugation between fluorenyl and terpyridine-ruthenium nitrosyl fragments on the ability of complexes to TPA ... 91
4.1.1 The synthesis of FCCT and FCC3T ligands ... 92
4.1.2 Synthesis of ruthenium-nitrosyl complexes with FCCT and FCC3T
ligands ... 98
4.1.3 Electronic spectra of [Ru(FCCT)(bpy)NO](PF6)3 and
[Ru(FCC3T)(bpy)NO](PF6)3. ... 101 4.1.4. The study of NO-release from complexes [Ru(FCCT)(bpy)NO](PF6)3 and [Ru(FCC3T)(bpy)NO](PF6)3 ... 106 4.1.5 The study of two-photon absorption of compounds
[Ru(FCCT)(bpy)(NO)](PF6)3 and [Ru(FCC3T)(bpy)(NO)](PF6)3 ... 108
4.2. Influence of the donating ability of the substituents in terpyridine ligands on the efficiency of ruthenium-nitrosyl complexes in TPA. ... 109
4.2.1. Syntheses of the ligands DMAFT, DMAFCCT and ruthenium-nitrosyl complexes based on them. ... 111
4.2.2. Dependence of the TPA cross-section on the donor strength of the substituent in a row of ruthenium-nitrosyl complexes with substituted
phenylterpyridines ... 118 CHAPTER 5. INVESTIGATION OF THE MECHANISM OF
NO-PHOTORELEASE FROM RUTHENIUM-NITROSYL COMPLEX WITH THE LIGAND 4'-(4-METOXYPHENYL)-2,2':6',2''-TERPYRIDIN ... 123
5.1 Synthesis and characterisation of the complex [RuII
(MeO-Phtpy)(bpy)(NO)](PF6)3 ... 123 5.2 Electronic spectrum and basic electronic transitions in the compound
[RuII(MeO-Phtpy)(bpy)(NO)](PF6)3 ... 126 5.3 NO-release from the complex [RuII(MeO-Phtpy)(bpy)(NO)](PF6)3 ... 130 5.4. Oxidation state of ruthenium within the release process ... 133
5.5. The by-product of the reaction of NO-release from the complex [RuII (MeO-Phtpy)(bpy)(NO)](PF6)3 ... 142 EXPERIMENTAL PART ... 145 PUBLICATION LIST ... 183 REFERENCES ... 185
ABBREVIATIONS AcOEt ethylacetate 2-acpy 2-acetylpyridine Alk alkyl bpy 2,2'-bipyridine ɧ-BuLi n-butyllithium
DFT density functional theory
dHFT 4'-(9,9-dihexyl-9H-fluoren-2-yl)-2,2':6',2''-terpyridine
DIPA diisopropylamine
DIBAL-H Diisobutylaluminum hydride
DMAFCCT (E)-7-(2-([2,2':6',2''-terpyridine]-4'-yl)vinyl)-9,9-dihexyl-N,N-dimethyl-9H-fluoren-2-amine DMAFT 7-([2,2':6',2''-terpyridine]-4'-yl)-9,9-dihexyl-N,N-dimethyl-9H-fluoren-2-amine DMF dimethylformamide DMP 1,1,1-Triacetoxy-1,1-dihydro-1,2-benziodoxol-3(1H)-one (Dess-Martin periodinane).
DMSO dimethylsulfoxide
EPR electron paramagnetic resonance equiv equivalent Et ethyl Et3N triethylamine EtOH ethanol FCCT (ȿ)-4'-(2-(9,9-dihexyl-9H-fluoren-2-yl)vinyl)-2,2':6',2''-terpyridine FCC3T 4'-((9,9-dihexyl-9H-fluoren-2-yl)ethynyl)-2,2':6',2''-terpyridine
FT 4'-(9H-fluoren-2-yl)-2,2':6',2''-terpyridine HOMO highest occupied molecular orbital
HPLC high-performance liquid chromatography HRMS high-resolution mass-spectrometry
in vitro in laboratory conditions
in vivo in living organism
IR infrared
LUMO lowest unoccupied molecular orbital Me methyl
MeOH methanol
MeO-Phtpy 4'-(4-metoxyphenyl)-2,2':6',2''-terpyridine
MGD N-methyl-D-glucamine dithiocarbamate
mp. melting point
NADP Nicotinamide adenine dinucleotide phosphate NLO nonlinear optics, nonlinear optical
NMR nuclear magnetic resonance
NONOate 1,1-R1,R2-2-hydroxy-2-nitrosohydrazine ɈɊȺ linear absorption (one-photon absorption) o-tolyl ortho-tolyl
Ph phenyl PhMe toluene
ppm point per million
py pyridine RNS reactive nitrogen species
ROS reactive oxygen species
rt room temperature
SCE saturated calomel electrode
SNP sodium nitroprusside
TD-DFT time-dependent density functional theory Tf triflate THF tetrahydrofuran TLC thin-layer chromatography TMS trimethylsilyl ɌɊȺ two-photon absorption UV ultraviolet ǻ heating at the boiling point
į chemical shift, ppm (NMR-spectroscopy)
INTRODUCTION
Last decades, since nitric oxide was proclaimed “Molecule of the Year” by the journal “Science”, many research teams has been aiming at in-depth study of physiological role of NO. Nowadays this compound is considered one of the main constituents in biological processes. The idea of a controlled nitric oxide release has become very attractive, for instance in order to be able to cause a selective influence on malignant cells. Indeed, depending on concentration, NO can inhibit the tumour growth or lead to the cell apoptosis. Exogenous NO-donors as modifiers of NO-levels in biological tissues are one of the most promising ones. S-nitrosothiols, organic nitrates, sydnonimines are practically important nowadays.
Especially interesting group of promising potent NO-donors is metal-nitrosyl complexes, in particular ruthenium-nitrosyls. The last ones are stable in aqueous media as opposed to the majority of alternative metal-nitrosyls. But the most important advantage of ruthenium-nitrosyl complexes is their ability to release NO under irradiation exclusively, that makes them perfect candidates as prodrugs for photodynamic therapy.
One of the targets for modern photodynamic therapy is the development of photosensitization methods of prodrugs in optical therapeutic window (700-1300 nm). Beyond this range of wavelengths the activation is ineffective due to a high extinction of electromagnetic waves intensity because of their absorption by biological tissues. Most of known photoactive NO-donors nowadays are able to release NO while being activated with waves of 350-600 nm waveband. Therefore the question of photosensitization of such compounds in vivo is a keystone for their future implementation in medical practice.
One of approaches, opening up the possibilities for photoinitiation of Ru-N bond dissociation in ruthenium-nitrosyl complexes in optical therapeutic window is two-photon absorption (TPA). This modern method has not been widely implemented in medical practice, however it is being thoroughly studied by scientists of different fields of science and technology – from medicine and microconstruction to 3D-data storage. High spatial selectivity of electromagnetic
waves influence on organism and reduced destructive effect (when employing IR-range waves) makes the local NO-release without damage dealt to adjacent tissues possible.
Revealing of main structural elements of ruthenium-nitrosyl coordination compounds capable of participating in two-photon absorption process and understanding the mechanism of NO-release process, is thereby an actual task in designing of promising NO-donors prodrugs.
CHAPTER 1. LITRERATURE OVERVIEW
1.1 Nitric oxide: molecular structure and general chemical characteristic. Nitric oxide (or nitrogen monoxide) is a colorless gas, which is monomeric at standard conditions; its molecules consist of an oxygen atom and a nitrogen atom connected by a multiple bond. Odd number of electrons in NO molecule makes the substance paramagnetic; in addition, it is the smallest thermally stable molecule-radical known. In terms of the molecular orbital method, it is logical to compare the structure of the compound with the structure of N2 molecule or with one isoelectronic to it – CO (figure 1.1).
Figure 1.1. A diagram of atomic and molecular orbitals of nitrogen and nitric oxide and schematic configuration of molecular orbitals
Appearance of an additional electron (in transition from nitrogen to nitric oxide), which is located on the antibonding ʌg NO orbital causes decreasing of the bond order from 3 (for N2 molecule) to 2.5 (for NO molecule). This has a corresponding effect on the nitrogen monoxide physical and chemical properties. In particular, bond length in NO molecule is 0.115 nm. It is an intermediate value between the length of the typical double bond N-O (0.120 nm) and the interatomic distance in ionized NO+ particle (0.106 nm), which bond order is 3.1 And vice versa reducing the compound to NO- increases the bond length to 0.126, which is caused by appearance of one more electron on the molecular antibonding orbital
and decrease of the potential is 9.25 eV; t compounds which is ca an electron from the an the following compou respectively. In practice be distinguished by an particle. Thus, for nitro is well over the one for – 1470 cm-1. 2
Realization of ele explanation of its coord following resonance for
Often, however, t dimerization, are mos structures:4
Analysis of the indicates strong intera molecule rotation; add of spin density is conce Due to little diff electronegativity the polar, its dipole momen Theoretical value has been obtained in th means of density func almost differ from the
bond force coefficient2. Nitrogen the value is well under the one char aused by significant stabilization of the ntibonding orbital. For comparison: ion unds: N2, O2, CO are 15.6 eV, 12
e, NO particle charge – neutral radical, nalyzing IR-spectra of the compound ozonium ion (NO+) vibration frequency
r nitric oxide – 1875 cm-1 – and, respec
ectron density distribution in nitric o dination behavior. In terms of the vale
rms could describe the structure of the
the compound properties, in particula st adequately explained in terms of
character of hyperfine splitting in action between unpaired electron ma
itionally, it indicates that 60% entrated on nitrogen atom.3
fference between the elements compound is rather slightly nt is 0.15D.
of the dipole moment, which he frame of this dissertation by ctional theory (DFT), does not
experimental one; it is 0.1 D.
Figu of el mole
monoxide ionization racteristic for similar e particle when losing nization potentials for .1 eV and 14.0 eV , cation or anion – can d, which contains the
y is 2377 cm-1, which ctively, for NO- anion
oxide is essential for nce bond method, the
compound:3
ar its low liability to Linnet’s “unpaired”
n NO EPR-spectrum agnetic moment and
ure 1.2. Distribution lectron density in NO ecule
Distribution of electron density in the molecule is shown on figure 1.2.
Being the simplest molecule with an odd number of electrons, nitrogen monoxide is a rather well studied compound from the point of view of its chemical properties. A great number of reactions between NO and radicals, atoms and other paramagnetic species became classical unique examples of reactions for kinetics study.
There are several number of commonly used laboratory methods of NO generation. Though the obtained nitric oxide as a rule contains impurities of other nitrogen oxides or volatile compounds. The most used method is reduction of dilute (~30%) nitric acid by copper:
8HNO3 + 3Cu = 3Cu(NO3)2 + 4H2O + 2NO
NO synthesized in this way is to be later purified in order to remove the impurity of nitrogen(IV) oxide by running the mixture of generated gases through aqueous solution of alkali. Another method of NO obtaining is reduction of alkaline metal nitrites:
2NaNO2 + 2NaI + 2H2SO4 = I2 + 4NaHSO4 + 2NO
2NaNO2 + 2FeSO4 + 3H2SO4 = Fe2(SO4)3 + 2NaHSO4 + 2H2O + 2NO An up-to-date but considerably more expensive method of obtaining NO in laboratory conditions is use of so-called NONOate-compounds. The method is used when controllable NO synthesis is essential, for example, in biological studies (see section 1.3.2)
In contrast to nitrogen, nitric oxide is an endothermic compound, therefore it is less stable. Even at a temperature slightly higher than the room temperature (30-50°C), but only under high pressure, nitrogen monoxide disproportionates with generating nitrogen(I) oxide and nitrogen(IV) oxide. Decomposition to the elementary substances is happening at significantly higher temperature – close to 7000 C in the presence of a catalyst.
Strong oxidizers can convert nitric oxide into compounds where nitrogen has higher oxidation states.5 The most typical nitric oxide reaction is interaction with oxygen, at which the compound is oxidized to nitrogen(IV) oxide:
2NO + O2 = 2NO2
Whereas interaction of NO with atomic oxygen, at which chemiluminescence is observed, was used for qualitative and quantitative determination of atomic oxygen:6
NO + O = NO2 + hȞ
Naturally, the presence of water in nitrogen monoxide and oxygen interaction changes the reaction path since NO2 itself can react with water. That’s why as a result of NO-oxidation by oxygen in humid condition nitrite-ions are generated. The reaction is commonly described by the following equation:7
4NO + O2 + 2H2O = 4H+ + 4NO2
-NO+ ion predicted by the theory indeed exists. Compound containing nitrosonium cation could be obtained in the reaction:
N2O3 + 3H2SO4 = 2NO+ + 3HSO4- + H3O+
Solidity at standard conditions of nitrosylsulfate acid NOHSO4 and similar compounds NO+ClO4-, NO+BF4 as well as their electrolysis mechanism corroborate an ionic nature of the compounds.5
1.2 Nitric oxide biological role
Number of published works devoted to nitric oxide biological role is rapidly growing during last four decades. As of February 2018, number of publications found in SciFinder search system were 196712. 0.4% of them were published before 1980, 22.4% - during 1981-1999, and the major part – 77,2% appeared as late as in the 21 century.
It was the discover of the role of this small molecule as a signaling molecule in cardiovascular and nervous systems that aroused so rapidly an increasing interest to it. In 1992 the journal Science called nitric oxide “Molecule of the year” and in 6 years, December 10, 1998 the Nobel prize for physiology and medicine was given to three researchers from the USA – Robert F. Furchgott, Louis G. Ignarro and Ferid Murad – for investigation of nitric oxide biological functions.
environment are often contradictory.8 For instance, nitrogen monoxide is the best example of a substance that demonstrates at the same time both cytotoxic and cytoprotective action.
A large number of chemical reactions characteristic for NO, in industrial or laboratory routine proceed quite slowly and thus requires using high temperature and pressure being at the same time thermodynamically disadvantageous9. From the other side, nitric oxide chemical transformations in biological environments take time from 0.002 to 2 s.10 These processes in NO biochemistry are usually divided into two types: direct and indirect. Direct effects caused by NO proceed quite fast and during them nitric oxide interacts directly with a biological target, whereas indirect ones are the results of NO interaction with oxygen or superoxide, that generates reactive nitrogen species (RNS), further interacting with biological objects. One of the advantages of such a differentiation is that the direct effects are caused generally by low NO concentrations ( 200 nM), while the indirect ones are the results of influence of significantly higher concentrations (! 400 nM).9
Nitric oxide principal biological functions are generalized on diagram
1.1.9,10,11
The main difference between direct and indirect processes is rate of its proceeding. High-speed reactions of immediate interaction between NO and biomolecules – mainly reactions with radicals or metalloproteins – are the direct processes.
A large number of NO main regulatory functions realize through its easy and rapid interaction with metalloprotein heme-prosthetic groups, such as guanylate cyclase (sGC), cytochrome P450, NO- synthase, hemoglobin etc. In such kind of processes the rate-determining step is diffusion of reacting molecules. Process of interaction between nitric oxide and Fe-S cluster protein aconitase proceeds considerably slower.12
Biomolecules having in their structure other metal ions, apart from Fe, are also susceptible to the influence of NO. This type of interaction could be represented by the vitamin B12 derivative, cobalamin, the nitrosylation of which leads to its reduced activity in the role of a cofactor of methionine synthase.
Reactions between nitric oxide and radical particles can have both positive and negative effects on cells. Lipidic and carboradicals are generated as a result of both oxidative stress and normal metabolism in a living organism. Nitrogen monoxide is able to attenuate effects caused by such reactive particles. For example, inactivation of tyrosyl radical generated in the process of ribonucleotide-reductase regular catalytic cycle by nitric oxide leads to inhibition of the mentioned enzyme.13 DNA synthesis suppression is an example of harmful effects of NO interaction with radicals in biological environment.12
But it is often that the reactions of NO are of the nature of “protective” functions for cells. NO is able to interact with radicals generated in the process of lipidic peroxidation – an important component of inflammatory processes and cell death – and so terminate the chains of reactive oxygen species (ROS) radical reactions, thus protecting the lipidic membranes:14
LOO· + NO· = LOONO L· + NO· = LNO
Macrophages protective functions in a living organism are also related to their ability to generate nitric oxide. Concentration of the latter in this case can considerably change depending on the “needs”. Due to the ability to vary NO concentration macrophages are able to participate in a wide range of processes aimed at organism protection – phagocytosis, cancer development suppression, tissues restoration, wounds healing etc.
As for NO indirect effects, they also can be divided into two categories – nitrosation and oxidation. The subjects of nitrosation are, first of all, thiol and aminogroups while oxidation is a process that refers to quite a number of substrates – from relatively easily oxidizable catecholamines and metal centers to compounds with a high oxidation-reduction potential, such as lipids, proteins DNA. As in the case of the direct effects, oxidation and nitrosation are regular cell metabolism processes but at the same time they can influence negatively generating substances toxic for cells.
Main RNS responsible for nitrogen(II) oxide indirect effects are nitrogen(III) oxide, N2O3, peroxynitrite, ONOO-, nitroxyl, HNO, and nitrogen(IV) oxide, NO2. Each of the particles depending on the redox potential, electrophilicity etc. interacts with certain biological substrates. Thus, the main nitrosating agent for amines and thiols is N2O3, which transforms them into nitrosamines and S-nitrosothiols, respectively.12 In turn, NO2 participates in oxidation processes. The molecule does not possess potential high enough for DNA oxidation15 but it can trigger lipidic peroxidation processes.16 Among the mentioned particles, peroxynitrite anion shows the strongest oxidizing ability. Under its influence, thiols, lipids, DNA can be oxidized, this particle also participates in the processes of tyrosine, guanosine and methionine nitration.16
Nitric oxide has a broad spectrum of effects on cancer growth – from triggering cell mutation to destructing malignant tumours. There are several reviews devoted to nitric oxide effects on the course of cancer.17,18 Numerous studies on the issue show that commonly nitrogen monoxide low concentrations in cancerous tumour, which is a result of permanent hypoxia, create so-called “safe
zone” and are a positive factor for cancer extension, promoting faster angiogenesis and metastasis (Figure 1.3).19,20 If, however, NO concentration changes in the direction of decreasing or increasing this leads to transferring to the “inhibition zone” or to the “kill zone”, respectively. Both concentration ranges are unfavorable for cancerous tumors and are potential targets in their treatment. Transferring to the very low concentrations range can be achieved by using NO-synthases (NOS) enzymes inhibitor compounds in therapy. The opposite approach is using nitric oxide donor compounds for NO concentration essential increasing, transferring to the “kill zone” and triggering cell death.
Generalizing the effects that NO can cause in the concentration range from 1nM to 1ȝM, we can say that NO low concentrations promote cells protection, their proliferation while high concentrations are responsible for blocking cell-cycle, apoptosis, aging.9 Besides, NO plays a central role in the cardiovascular system as an endothelium-dependent relaxing factor,21 in the central nervous system it is involved in the nerve impulses transmission and memory processes, and also performs a regulatory function in the cerebral circulation.
Having chosen nitric oxide as such a multifunctional biological regulator, nature provided living organisms with mechanisms of its biosynthesis.
Figure 1.3. Cancerous tumour reaction to nitric oxide at its different concentrations
1.3 NO generation from organic compounds
1.3.1 Natural pathway of nitric oxide production
Nitric oxide is one of the most universal molecular regulators of cellular metabolism. The main way of its synthesis in living organism is oxidation of the amino acid L-arginine in the presence of the NOS enzymes. The conversion occurs in two steps (Scheme 1.2).22
Scheme 1.2. Enzymatic transformation of L-arginine into L-citrulline with the formation of NO molecule
Initially, the 2-electron oxidation of L-arginine and its hydroxylation in the presence of oxygen and NADPH23 leads to the formation of NȦ -hydroxy-L-arginine (NOHLA). Tetrahydrobiopterin accelerates the course of this step acting as a cofactor.24 Further NOHLA oxidation (the next transformation step) leads to the formation of L-citrulline and one molecule of nitric oxide. The general scheme of the conversion of L-arginine to L-citrulline can be described by the following equation:
2L-arginine + 3NADPH + 3H+ + 4O2 = 2L-citrulline + 2NO + 4H2O + 3NADP+ Moreover there are four other cofactors, the absence of which either slows the reaction or makes it impossible to pass.25 The NOS enzyme group includes three main enzymes - endothelial NO synthase (eNOS), neuronal NO synthase (nNOS), and inducible NO synthase (iNOS); the fourth type - mitochondrial synthase (mtNOS) is identical with the Į-form of nNOS.25 The first two enzymes are calcium-dependent and are activated in the presence of high concentration of calcium ions by calcium-calmodulin complex; typically, their activity period does not exceed few minutes after stimulation. Activated eNOS and nNOS are involved
in the conduction of nerve impulses, peristalsis regulation and instantaneous regulation of blood pressure.26 Although all three forms of NOS require a calcium-calmodulin complex, iNOS exhibits a much higher affinity for it, which leds to longer activity period (up to days) and lower calcium-calmodulin complex concentration required.25 iNOS activation is the response to the internal stimulation with a variety of microorganisms metabolic products, some cancer cells, cytokines etc. Activated enzymes produce millimolar amounts of NO (interacting with ROS – peroxynitrite), showing an immune-protective function by oxidizing proteins and DNA.26 However, in the lack of oxygen (i.e. chronic inflammatory processes), the level of RNS can be significantly lower, and the nitric oxide provokes mutagenesis and angiogenesis instead, thus connecting directly inflammation and the initiation of cancer.25
Considering such a large number of biological processes in which nitric oxide is involved and which in its absence or its low concentration slow down, stop or activate undesirable chemical reactions causing a number of diseases, it seems attractive to obtain a mechanism for regulating the concentration of NO in living organism.
1.3.2 Classical donors of nitric oxide based on organic compounds.
Nitric oxide donors or NO-donors is a group of compounds or functional fragments of supramolecular systems that can release nitrogen monoxide when exogenously applied.27 Lack of NO in a particular place of a living organism can trigger a number of pathological processes that will manifest themselves as symptoms of various diseases. Although free nitric oxide is used successfully for medical purposes (locally, for the treatment of dermatitis,28 or in the form of inhalations in pulmonary hypertension in newborns29), the use of NO-donors can significantly increase the range of possible exogenous effects on the body.28 Chemical stability and the capability of local NO elimination of NO-donors allow
the controlled delivery of nitrogen monoxide to various parts of the living organism.
NO-donors can be divided into several groups depending on the method of NO-release activation. According to the classification proposed by Philish and Stamler,30 three major groups can be distinguished: 1) NO-donors that liberate NO spontaneously; 2) NO-donor that release NO after chemical interaction with acids, alkalis, metals, thiols; 3) NO-donors that release nitric oxide after their enzymatic oxidation (Scheme 1.3).
Scheme 1.3. Classification of NO-donor by the initializing of the NO release.
In this work we propose to sort out one more type of donors – compounds that are able to release NO under their photochemical activation, since many studies and research papers published recently are devoted to photoactive compounds that are amenable to NO release.
O O HO
ONO2
Organic nitrates are the most widespread example of NO-donors. Since they were the first approved drugs used for NO-level control they are worthy of special attention. Organic nitrates are esters of alcohols (polyols in general) and nitric acid. Glyceryl trinitrate (GTN) is the most thoroughly studied compound of this class (Figure 1.4). Its use is not limited to vasodilating effects on the vessels. Among the pathologies for the treatment of which the use of GTN is approved, one can mention cardiac dysfunctions31, hypotension and headaches,32 angina and cancer.33 Other drugs used are isosorbide dinitrate (ISDN), pentaerythril tetranitrate (PETN), isosorbide-5-mononitrate (ISMN) (Figure 1.4).34 The mechanisms by which the nitric oxide is liberated from organic nitrates are still contradictory. However, the proposed schemes include the stage of interaction with one of the enzymes: glutathione-S-transferase, cytochrome P450, xanthine oxidoreductase, aldehyde dehydrogenase.35
Glycerinetrinitrate (GTN) Pentaerythritol tetranitrate (PETN) Isosorbide dinitrate (ISDN) Isosorbide-5-mononitrate (ISMN) Figure 1.4. The main representatives of the class of organic nitrates
The use of organic nitrates can lead to a number of side effects,34 since these drugs show their effects in the body in whole, without specific localization. However, the most significant disadvantage of using organic nitrates is the tendency of the body to rapidly developed tolerance to these compounds.36
This feature of organic nitrates can be very useful in the treatment of cancer. The growth of cardiovascular tolerance to GTN allows its use in complex cancer therapy, along with cytostatic drugs (i.e. doxorubicin) without systematic side effects from continuous high NO levels. Research on the use of such a combination
ONO2 O2NO ONO2 ONO2 O2NO ONO2 O2NO O O O2NO ONO2
in the treatment of mice prostate cancer shows a more effective inhibition of tumor growth than in the treatment of exclusively doxorubicin.37
Other commonly used compounds in cancer treatment studies are nitrozotioles, diazeniumdiolates (NONOate) and sydnonimines (Figure 1.5). S-nitrosothiols are compounds with a nitroso group which is directly linked to the sulfur atom. Most compounds of this class are unstable, but several of them are sufficiently stable and have already proven themselves as active drugs (i.e. S-nitroso-N-acetylpenicillamine (SNAP)) (Figure 1.5).32
S-nitroso-N-acetylpenycillamine (SNAP) 3-morpholine-sydnonimine hydrochloride (SIN-1) Sodium (Z)-1-(N,N- diethylamino)diazen-1-ium-1,2-diolate
Figure 1.5. Representatives of other classes of NO-donors used in oncology practice
The main advantage of S-nitrosothiols is better biocompatibility compared to other NO-donors, in particular NONOate. This conclusion comes from the fact that nitrosothiols are naturally present in biological systems, as for instance S-nitroso-L-cysteine (CysNO) or S-nitroso-L-glutathione (GSNO).38 The mechanism of NO-release from S-nitrosothiols is strongly dependent on the external conditions (Scheme 1.4).
Scheme 1.4. Mechanisms for the release of nitric oxide from S-nitrosothiols O N N N O H2N + Cl -O N S O OH HN O N N N O -O Na+
Despite the large number of publications devoted to the study of these compounds in vivo, they have not yet found direct application in medicine. Their ability to release NO in different environments, the ability to release it in the form of a radical or nitrosonium-cation, predictable pathway of metabolic degradation, and an almost complete absence of tolerance development with prolonged use – all these benefits make S-nitrosothioles compounds of interest as nitric oxide donors in medical practice.39
Another class of NO-donors is diazeniumdiolates (or NONOates). These compounds are known since the 1960s, when its first DEA/NO agent was synthesized for the first time.40 However, they became the object of numerous studies only in the 90s of the last century,39 after the biological role of nitric oxide had been discovered. In their structure diazoniumdiolates contain [N(O-)N=O] group. Considering the atom the group is bound to, NONOate are divided into S-linked, O-S-linked, N-S-linked, or C-linked. However, only Nitrogen-bound NONOates play significant role in biology.38,41
Under physiological conditions (pH 7.4 and temperatures of 37 °C), compounds of this class undergo acid hydrolysis producing 2 equivalents of nitric oxide per diazeniumdiolate group:32,41
Spontaneous NO release, no need for additional activators of this process (i.e. thiols, metal complexes or enzymes), first order reaction rate moves diazeniumdiolates in one row with S-nitrosothiols and sydnonimines of promising NO-donors.
Easy predictable NO-liberation rate and its final concentration are important qualities of NONOate, due to which compounds of this class are widely used in studies aimed at desing of anticancer drugs.27 On the other hand, the main advantage easily transforms into the serious drawback when using this compound as drug – NO-donors based on NONOate can release NO before reaching the main
target. The use of targeted drug delivery can help to overcome this disadvantage, although it significantly complicates the drug preparation process.
One of approaches of targeted delivery of this class of compounds is demonstrated in the paper of Wang and coworkers.42 Using one of NONOates – PYRRO/NO compound as NO-donor, the researchers inactivated the compound by binding it with a certain amino acid sequence through the oxygen atom of the diolate group. The use of amino acid sequence of one segment of the substrates of prostate-specific antigen (PSA) allowed making modified PYRRO/NO compound sensitive to the presence of this enzyme. PSA is an enzyme produced by both healthy and cancerous cells of the prostate epithelium in significant amounts.42 Increased concentration of this enzyme is observed in metastases of prostate cancer.
The resulting PYRRO / NO based drug must be inactive before hydrolysis that occurs in the presence of PSA or Į-chymotrypsin. As a result, spontaneous release of NO in vitro from the resulting compound was observed only in the presence of one of these enzymes in solution (Scheme 1.5).42
Scheme 1.5. Nitric oxide release from peptide-bound PYRRO/NO by enzyme activity PSA
Another class of NO-donors already used as medicines are sydnonimines. Sydnonimines are mesoionic heterocycles, which include the 1,2,3-oxadiazole ring and contain an imino group in the 5 position. For the first time, independently of each other, compounds of this class were synthesized in 1957 by Brooke43 and Ohta.44 Under normal conditions and in the absence of light sydnonimines are stable solids.
Sydnonimines release nitric oxide in an alkaline medium and the passage of this process is greatly facilitated by the presence of oxygen or under excitation with UV irradiation. The most studied example of this class is
O O Peptide O N N N O -+ -O O N N N O -+ -O N N N O -+ NO PSA -RCO2 -H2CO
3-morpholinosydnonimine (SIN-1, linsidomine) (Figure 1.5). The medicinal product under the brand name "Molsidomine" is based on this compound. The release of NO from this product is shown in Scheme 1.6.45 The first step of decomposition is the enzymatic deacetylation of molsidomine in the liver with the formation of linsidomine. At physiological or alkaline pH linsidomine undergoes a non-enzymatic cycle opening. The formed product is completely stable at pH 7.4 in absence of oxidants, but even small amounts of oxygen lead to the formation of radical cation which decays producing nitric oxide and N-morpholino-aminoacetonitrile.
Scheme 1.6. NO-release from medicinal product "Molsidomine"
Typically sydnonimines donate NO in peroxynitrite anion form.44 As shown on the Scheme 1.6 during the molsidomine transformation the stoichiometric amount of the superoxide radical is formed, and since the interaction of nitrogen monoxide with the superoxide radical is extremely rapid and the diffusion is the only limiting process, the formation of toxic peroxynitrite is inevitable. This feature of sydnonimines, of course, reduces the spectrum of their possible use in medicinal purposes. The advantage of these compounds is that addiction to them does not appear, and a tolerance to sydnonimines does not arise even with prolonged use.
1.3.3 Nitrobenzenes as examples of photoactive NO-donors
The ultimate goal of most studies of nitric oxide donors is the compound that is capable of controlled and predictable local NO-release. This substance would be
able to give a clear answer to the question of how different concentrations of NO affect different biological objects and to allow finding the optimal rate and the required concentration of nitrogen monoxide for a particular physiological effect.46 The use of a controlled NO-donor will reduce the dose required for treatment and reduce the side effects associated with the release of nitric oxide beyond the immediate biological target.47 It is clear that controlled release of NO is also extremely important for the effective treatment of cancer with the use of NO-donors, since it significantly reduces the toxic effect of RNS on healthy cells of the body.48 Another potential use of materials capable of controlled NO-release is the creation of various devices, such as artificial blood vessels or tubes for hemodialysis.49 One of the main ways of obtaining of such compounds is the design of photoactive NO-donors.
Among the substances that are capable of photoinduced release of NO, the most promising and studied are derivatives of nitrobenzene and metal nitrosyl complexes. However, a large number of works is also devoted to the study of supramolecular ensembles (i.e. liposomes, micelles), nanoparticles and polymer materials that are capable of releasing or activating the NO-donors, which in turn can produce nitric oxide locally.27,41,50,51,52
The investigation of aromatic nitrocompounds as NO-donors was started by Sortino and Miyata groups. In general, the functional group that directly acts as a donor of NO in these compounds is an
aromatic ring with a nitrogroup and bulky substituents in the ortho-position (i.e. CH3, CF3, Ar etc.) (Figure 1.6a).27 Due to the presence of closely located substituents, the nitrogroup is forced to accept the position almost perpendicular to the plane of the aromatic ring, loosing the conjugation with
the benzene ring.53 In this arrangement, the p-orbital of the oxygen atom considerably overlaps with the orbitals of the aromatic ring, both in the main and
ɚ b Figure 1.6. Nitrobenzene-based NO-donors NO2 R R' R = CH3, CF3, Ar R' = H, CH3, CF3, Ar F F F N O O
excited states. In this view the process NO-release from o-substituted derivatives of nitrobenzene occurs, probably, through the stage of isomerization of the nitro into the nitrite derivative and the subsequent decomposition of the latter into NO and the phenoxy radical due to O-N bond breakage (Scheme 1.7).54 According to the authors, the key factor for the possibility of isomerization is the orthogonal arrangement of the nitrogroup and the benzene ring.55,56
Scheme 1.7. The mechanism of nitric oxide release from an anticancer drug flutamide
Nitrobenzene-based NO-donors are thermally stable under physiological conditions, and the release of nitric oxide from these compounds can be controlled by electromagnetic irradiation. Also, the advantage of nitrobenzenes as photoactive NO donors is the absence of metal ions, the presence of which could itself provide cytotoxic properties. However, the significant disadvantage of this class of NO-donors is the need to use electromagnetic radiation of UV-region, which is significantly absorbed by biological tissues and fluids and is hazardous to healthy cells.
An example of work aimed at overcoming this lack of nitrobenzenes is the work of Naoki Miyata group.55,57 Modification of the starting 2,6-dimethylnitrobenzene (which is responsible for the NO release) by the introduction of a compact fluorophore (such as 7-hydroxycoumarin or 7-aminocoumarin) made it possible to shift the band of absorption of compounds from UV to the visible domain of electromagnetic waves (Figure 1.7a and 1.7b).57 An interesting fact was that in the case of a DEAMC-DNB in which there is a complete conjugation of the aminogroup with the nitrogroup through the ʌ-chain, release of nitric oxide is not
observed under the irradiation of compound at 430-460 nm. Owing to the efficient intramolecular charge transfer, the bond order of C-N(NO2) bond increases to double, and nitro-nitrite isomerization becomes impossible. However, in the case of a compound where the ʌ-system cross-conjugation is present (Bhc-DNB), the irradiation results in NO-release. Under excititation of the compound no significant
a O Br HO O NO2 O N O NO2 Bhc-DNB DEAMC-DNB b
Figure 1.7. New compounds based on 2,6-dimethylnitrobenzene (a) and their electronic spectra (b).
charge transfer occurs in the molecule (as could be concluded from the absence of an intense band in the UV-visible spectrum) and the band 350-430 nm appears due to the absorption of coumarin fragment, which acts as an antenna. Thus the researchers were able to shift the excitation wavelengths required for nitrobenzene activation from UV to visible domain.57
Another elegant way to avoid the UV irradiation to initiate NO-release from nitrobenzenes is the use of two-photon absorption method. This approach allows shifting of the excitation wavelength up to the infrared (IR) region, which does not show a deleterious effect on the body and is more permeable for biological tissues.
Binding of 2,6-dimethylnitrobenzene through a styryl and weakly conjugated amide fragment to fluorescein (which is famous for its ability to produce two-photon excited fluorescence58) researchers obtained a Flu-DNB compound (Figure 1.8) that released nitric oxide when irradiated with an IR pulse laser with a wavelength 720-800 nm.
Figure 1.8. Flu-DNB is a compound capable of releasing NO by two-photon photo-excitation in wavelengths of 720-800 nm. NO2 O H N O O HO COOH
1.4 NO-donors based on coordination compounds
1.4.1 General characteristics of nitrosyl complexes.
Today metal-nitrosyl complexes are considered as promising objects for targeted delivery of nitric oxide.59 They are especially interesting as photoactive NO-donors. Before consideration of the particular examples of the NO-donor compounds one should first concentrate on several aspects of the coordination behavior of nitric oxide.
The most typical and common way of coordination of nitric oxide to metal is a monodentatable type of binding, in which the nitrogen is a donor atom. Upon such coordination, two types of geometry are possible depending on the angle M-N-O – straight and angular. The differences between them can be explained by the valence bond and orbital hybridization theories.
Considering that straight or almost straight geometry of the complex is a result of the sp-hybridization of nitrogen orbitals, it is assumed that the nitric oxide in such compounds is in the form of NO+; while the angular geometry requires sp2
-hybridization, and in this case NO is in the form of NO-. Resonance forms, whose combinations could describe the linear and angular geometry, are shown in the Scheme 1.8.2 Those resonance structures where radicals are present can be considered as forms that possess a lone pair of electrons, and the corresponding complexes accept an intermediate geometry between the linear and angular, which is characteristic of the nitroxyl complexes.
The formation of nitrosonium-containing compounds requires the transfer of one electron from nitric oxide to a metal center, followed by ı-binding and ʌ-backbonding interaction dM ĺ ʌ* (Figure 1.9), and the formation of complexes that could be described as those containing nitroxyl anion is preceded by the transition of the electron from metal to the nitric oxide (Scheme 1.8).60
Scheme 1.8. Resonance forms of linear and angular geometry of metal-nitrosyl complexes
Nitrosonium cation NO+
Nitric oxide NO· Nitroxyl anion NO
-Figure 1.9. Simplified schemes of molecular orbitals for straight and bent geometries of mononitrosyl complexes M O M N O N M N O M O N M N O M N O Nitrosonium cation Nitroxyl anion Nitric oxide ı-bonding ʌ-dative interaction ı-bonding ı-bonding ʌ-dative interaction ʌ-dative interaction M N O M N O M N O
Thus, in the linear coordination form the oxide acts formally as a donor of 3 electrons, whereas in the angular form it is a donor of 1 electron. Both the method of valence bonds and the method of molecular orbitals showed that the increase in the electron density at the M-N bond due to the backdonation leads to the increase of this bond’s order though by reducing the energy of N-O bond.
Due to the discovery of the biological role of nitrogen (II) oxide, the interest in the coordination compounds of this molecule with metals has exponentially increased since the 1980s. Table 1.1 presents a general information about all nitrosyl complexes found in the Cambridge Structural Database (CSD) in 2014.60
Angle (°) Number of structures Geometry Example 180-160 2519 Linear [Mn(NO)(CO)4]61 160-140 121 Intermediate [Ir(NO)2(PPh3)2]62 140-100 115 Angular [Co(NO)(S2CN(CH3)2)2] Table 1.1 Generalization of metal-nitrosyl complexes from the CSD database in 2014
There are complexes (absent in the table above) that contain metal atoms bound both to oxygen and nitrogen atom of the NO group. Total number of such complexes is small; usually they are unstable and could form as a result of photoexcitation of nitrosyl complexes as transitional states. The particular example of such compounds is [Ni(NO)(Ș5-Cp*)], where Cp - pentamethylcyclopentadienyl anion. The Ni-N distance in it is only 0.4 Å shorter than the Ni-O distance, and the Ni-N-O angle is 93°.63
Apparently, the vast majority of compounds have a linear M-N-O fragment. Complexes in which this angle is much smaller are basically the complexes of the right half of d-metals. An explanation for this lies in the presence of bigger number of electrons at the d-sublevel that reduces the ability of metals to accept three electrons from nitric oxide. But only in this case the linear geometry of the complex is possible.1
Among the compounds found in CSD, there are also those containing from 2 to 4 NO-fragments that can assume either the same or different geometry.64 For example, in the compound [RuCl(NO)2(PPh3)2]+ (Figure 1.10)65, both atoms of one of the nitrosyl ligands and the metal atom lie on one straight line, while the other nitrosyl ligand is bound with an angle substantially less than 180°.
As shown in Figure 1.10, the lengths of the Ru-N and N-O bonds are somewhat larger in bent conformation, indicating a weakening of these bonds compared to a linear Ru-NO fragment.
Nitric oxide in linear conformation in complexes is a very strong ʌ-acceptor. The use of photoelectron spectral methods allowed to assess the ability of NO and other ligands to withdraw electron density from metal atoms through the ʌ-backdonating interaction.66 Among other ligands, nitrosyl cation occupies leading positions. In particular, its ʌ-withdrawing ability is the highest in the following series:
NO > CO > Ș2 - MeCŁCMe > H2C=CH2 > SO2 > N2 > PMe3 > SiCl3 > NH3 > CN > H > Cl > SiMe3 > CŁCH 66
Due to the significant shift of the electron density, the M-N distance significantly decreases with increase of dM ĺ ʌ* dative interaction. For example, in complexes that contain both linearly coordinated nitric oxide and ammonia as ligands, the M-NNO bond length is less than the bond length of M-NNH3 by 0.35-0.4 Å. The values of the M-N bond lengths in complexes indicate that these bonds are covalent for the most part. In view of this, the description of complexes as ionic compounds of nitrosonium-cation or nitroxyl anion becomes less relevant.
Another approach of describing complexes with non-innocent (redox-active) ligands (one of which is nitric oxide) is the Enemark-Feltman notation. Its advantage is the omitting of the concept of oxidation states of the central atom and ligands. Figure 1.10. Structure of cation
[RuCl(NO)2(PPh3)2]+ (the phenyl
When utilizing the Enemark-Feltman formalism for complexes with nitric oxide, the latter are designated as {M(NO)x}n, where x is the number of NO ligands and n is the total number of electrons located on the d-orbitals of the metal atom and ʌ* – orbitals of nitric oxide. It is assumed that the metal atom is in its formal oxidation state, and nitric oxide is neutral.64 Examples of the use of the Enemark-Feltman notation for coordination compounds nitric oxide are shown below:
[Co(NH3)5NO]2+ CoII d7 {Co(NO)}8
[Fe(SR)2(NO)2]- FeI d7 {Fe(NO)2}9 [RuCl2(terpy)(NO)]+ RuIII d5 {Ru(NO)}6
[Mn(CN)5NO]3- MnII d5 {Mn(NO)}6
(terpy – terpyridine)
Although the description of NO-complexes in this way eliminates ambiguity in determining the oxidation state of central atom and ligands, in some cases assigning a charge to them helps in understanding of electronic and magnetic properties of compounds.
To complete a picture of the coordination capabilities of NO, its ability to act as a bridging ligand should be considered as well. Similarly to the coordination behavior of carbon monoxide, nitric oxide is capable of binding two, three, and in some cases four metal atoms, stabilizing metal cluster complexes (Figure 1.11). An example of such a cluster is homobimetallic nitrosyl copper complex [(Cu2(XYL-O- )(ȝ-NO)](PF6)2, where XYL-O is 2,6-bis(bis(2-pirydyletyl)-aminomethyl)phenolate) (Figure 1.11a). This compound is interesting because of the fact that ineracting with
a b c
Complex cation67
[(Cu2(XYL-O-)(ȝ-NO)](PF6)2
Claster-type nucleus
Ru3Pt(ȝ4-NO) 68
The structure of the cluster 69
[{Re3(ȝ-H)3(CO)10}2(ȝ4-Ș2-NO)]
-Figure 1.11. Some bridging-coordination types of nitric oxide N py O Cu py N py Cu py N O 2+ Ru Ru Ru N O Pt N (CO)3 Re (OC)4Re Re (CO)3 O Re (CO3) Re(CO)4 (CO3) Re
nitrite anion and transforming into [(Cu2(XYL-O-)(ȝ-O)]+ it emulates the nitrite reductase activity (an enzyme consisting of diatomic copper cluster). Indeed, it is known that the main mechanism of this transformation includes the copper-nitrosyl complex as an intermediate stage.70
1.4.2 Study of metal-nitrosyl complexes by means of IR spectroscopy
Modern physicochemical methods allow an accurate characterization of metal-nitrosyl complexes; in particular, it is possible to determine the binding parameters and electronic structure of the M-N-O fragment without X-ray diffraction analysis. However, the interpretation of the obtained spectral data should be carried out with extreme caution and captiousness. In cases of possible ambiguity in the interpretation of certain results, methods of quantum chemistry serve as an auxiliary tool and enable a deeper understanding of the electronic structure of the complex.
The most important and frequently used spectral methods in the study of nitrosyl complexes are IR, EPR, UV and NMR spectroscopy. Less commonly used and more specific methods are the Mössbauer spectroscopy, X-ray (XAS) spectroscopy, magnetic circular dichroism, and the study of magnetic susceptibility.71
The use of vibrational spectroscopy for the study of nitrosyl complexes has become widely used. In fact, the dependence of the characteristic vibrations of NO on the donor-acceptor properties of the environment can serve as a tool for understanding the nature of the interaction of metal atom with nitric oxide. As indicated in section 1.1, the valence vibrations of free NO radical have a frequency of 1875 cm-1. After coordination of NO to the metal atom, the value may either increase or decrease depending on different parameters such as the nature of other ligands in the coordination sphere, the electronic configuration of the central atom, the total charge of the complex and its geometry. For metal-nitrosyl complexes there is a general tendency for the increase of vibrations frequency with the increase of the positive charge of nitrosyl ligand from 1300 cm-1 to 1950 cm-1.2 In addition, for compounds with linearly coordinated nitric oxide this value lies within the limits of 1950-1450 cm-1, and for those in which the M-N-O angle is considerably less than
180° vibrations in the region of 1720-1400 cm-1 are more typical; for bridging nitric oxide – from 1650 cm-1 to 1300 cm-1 (Figure 1.12).
Figure 1.12. Frequency ranges of valence vibrations of the N-O bond depending on the type of nitric oxide coordination
As shown, these ranges strongly overlap and an unambiguous determination of the coordination type on the basis on the value of the frequency is not possible. Nevertheless, if the frequency value is higher than 1750 cm-1, the linear geometry can be accurately established.
More correct and reliable correlation can be considered for the complexes similar in their electronic structure. For example, if the central atom is represented by one of the transition elements of 8th group (Fe, Ru, Os), and the complex have the following structure {M(NO)}6-8 (by the Enemark-Felthman notation) (Table 1.2).71
Complex Frequency {M(NO)}8 1270-1500 {M(NO)}7 1600-1700 {M(NO)}6 1800-1960 Table 1.2. Valence vibrations of N-O in complexes of metals of the 8th group metals.
As it was mentioned, the frequency of the valence vibrations of nitric oxide in complexes depends on many factors (in particular, on the nature of other ligands present in the complex). In the work of De La Cruz and Sheppard, authors made an attempt to summarize the effects of other ligands on the variation of the frequency of the vibration of the N-O bond.72 A brief summation is presented in Table 1.3.
Charge and ligands ǻȞ(NO) (cm-1) Ⱥ. Ionic charges
Unit of positive charge +100 Unit of negative charge -145 B. Electron-withdrawing ligands
Linear cyanide +50
Linear halogens +30
Bridging halogens <+15 C. Linear or bridging CO ligand § 0 D. Electron-donating ligands
PF3 +10
P(OPh)3 -30
PAlk3 (Alk – alkyl) -70
Ș5-C5H5 -60
Ș5-C5H4Me -70
Ș5-C5Me5 -80
Table 1.3. The influence of ligands of different nature and charges on the vibrational frequency of nitric oxide in
complexes.
1.4.3. Ruthenium-nitrosyl complexes as NO donors
The idea of utilizing of metal complexes as NO-donors falls in line with the fact that the primary goal for the attack of NO in the human body is iron-containing enzymes.32 Among the coordination compounds with nitric oxide donating ability sodium nitroprusside (SNP) is the most studied and well known one. More than for 90 years SNP is used for medical purposes as a hypotensive drug of the class of vasodilators. In the solid state Na2[Fe(CN)5NO]·2H2O can be stored for years in the absence of light and moisture, but in the solution the compound becomes extremely photosensitive and reactive to oxygen.44
The mechanism of NO release from SNP in vivo is not yet fully understood, but this process is thought to be initiated by photoexcitation or one-electron reduction, and the presence of thiols accelerates it (Scheme 1.9).44
Scheme 1.9 Possible mechanisms for the release of nitric oxide from SNP
As can be seen from the scheme, the process of reduction and subsequent decomposition of SNP is accompanied by the release of cyanide anion (sometimes up to 5 equivalents in biological conditions)44; this can lead to significant toxic effects on the whole body if significant amount of SNP was used. Also, the spontaneous decomposition of the compound increases the local concentration of the cytotoxic peroxynitrite anion. But, although SNP has some deleterious effects, its use as a vasodilating agent is justified, since the use of even small amounts of the drug is often enough to provoke biological effects. However, the use of this compound is very limited, and the search for new cooordination compounds that would be devoid of these shortcomings is a topical issue.
Among other metals, ruthenium attracts attention due to its ability to emulate the coordination features of iron.2 Unlike the latter, the ruthenium complexes are always low-spin ones, both in the oxidation states +3 or +2, which contributes to their higher stability compared to iron complexes, as well as the lower lability of the ligands (including nitric oxide) in its coordination environment;59,73 this is a prerequisite for their biological application. Confirmation of this can be a found in comparison of SNP with its Ruthenium analogue [Ru(CN)5NO]2-. Like sodium nitroprusside, [Ru(CN)5NO]2- is capable of being excited with electromagnetic waves, but unlike SNP, this compound does not release toxic cyanide ions in solution.70
Ruthenium-nitrosyl complexes also have several advantages, which are often not typical for coordination compounds of other metals - good water solubility,
resistance to air oxidation and low cytotoxicity.74 The kinetics of ruthenium interaction with bioligands is often compared to the effects caused by platinum on the body;75 however, numerous studies indicate a significantly lower toxic effect of ruthenium, which may be due to its close to iron coordination behavior and its ability to bind albumin molecules and iron-transporting transferrin proteins.76
Depending on the type of ligands in the coordination sphere, ruthenium can exhibit several basic oxidation levels that can exist in the biological environment – Ru(II), Ru(III) and Ru(IV). In the presence of reducing agents as glutathione or ascorbate Ru(III) could easily be reduced to Ru(II). Coordination compounds in which ruthenium is in the oxidation state +2 are more susceptible to ligand exchange and readily interact with N-donor or S-donor biomolecules.44 By changing the donor-acceptor properties of the ligands (i.e. their ability to ʌ-binding) the energy and strength of the bond Ru-NNO in the ruthenium-nitrosyl complexes can be varied affecting the efficiency and rate of nitric oxide release.44
An important advantage of ruthenium (along with Mn and Fe) in comparison with other metals is the photoactivity of its nitrosyl complexes. Despite the high stability of the coordination compounds of ruthenium with nitrogen monoxide, they are capable of releasing NO under irradiation by electromagnetic waves of a certain length.48 Due to this feature ruthenium-nitrosyl complexes are intensively studied for the purpose of obtaining the NO-donor capable of local, time-controlled, photosensitive release of nitric oxide.
The first studies of NO-release from ruthenium-nitrosyl complexes under the influence of light were carried out on {Ru(NO)}6 compounds with monodentate ligands – mainly amines and halogens (Figure 1.13).
The investigation of nitric oxide photorelease and the nature of corresponding photoproduct was carried out in 1983 by Sinitsyn and his collaborators; in this case the compound K2[RuCl5(NO)] was chosen as NO-donor. The signal observed in EPR spectroscopy during the experiment of photorelease having g-factor equal to 2.34 denoted the presence of paramagnetic low-spin d5-Ru(III) particles in the solution.77
The anion of
K2[RuCl5(NO)]
RuNOCl3 Cation of
trans--[Ru(NH3)4(X)(NO)]3+
Figure 1.13. Examples of the photoactive {Ru(NO)}6 complexes
with monodentate ligands
The stoichiometric ratios confirmed the previously proposed NO-release mechanism:
Ru-(NO) + solv ĺ RuIII-(solv) + NO
The quantum yields (ij) found for compounds K2[RuCl5(NO)] and RuNOCl3 are 0.06 and 0.012 respectively.78 This difference in the values for two quite similar compounds arises from the fact that the presence of a greater number of negatively charged ligands (5 chlorine atoms per ruthenium atom in the first compound against 3 chlorine atoms in the second) facilitates the process of NO-photorelease. Today these compounds are well studied and are commercially available.77
Studies of analogous compounds in which chlorine atoms in the equatorial plane are replaced with ammonia molecules – trans-[Ru(NH3)4(X)(NO)]3+ – have shown that the ligand that is in the trans-position to nitric oxide has a significant effect on the rate and efficiency of NO-release. Thus, in the case of the phosphite P(OEt)3 ligand the quantum yield of NO-release was ij = 0.3, while for the compound with the imidazole ligand in the trans-position to NO this value was only 0.04.77
Giving preference to polydentane ligands over monodentate ones in the study of compounds for the purpose of their further use for medical purposes is determined by a greater stability of chelating complexes and complexes with macrocyclic ligands.77 The necessity of using complexes with high stability constants becomes clear if we take into account the infinite number of biomolecules in the body that can strongly bind metal atoms, competing with monodentate ligands. Thus, polydentane
H3N H3N NH3 NH3 Ru NO X X = NH3, py, 4-acpy, P( OEt)3 3+ Cl Cl Cl Cl Ru NO Cl
2-ligands make it impossible to bind metals with bio2-ligands, thus reducing or eliminating the harmful effects of the former;77 and also, which is important for photoactive compounds, polydentate ligands provide the stability of complexes, and therefore thir photochemical characteristics.
Since prosthetic groups of heme proteins (hemoglobin, sGC, cytochromes) are the primary targets for nitric oxide attack in the body (see section 1.2), one of the first attempts to receive NO-donor based on coordination compounds of ruthenium with polydentane ligands was made precisely with the use of porphyrins.73 The resulting compounds of the general formula [(P)Ru(NO)Cl], where P is porphyrin, were thermally more stable and less susceptible to oxidation by oxygen in the absence of irradtiation than similar iron complexes. However, the reverse recombination of released NO molecule was too fast (k § 1*108 M-1s-1), that made these compounds unsuitable for targeted NO delivery.48,73,79
More promising from a practical point of view are non-porphyrin-type ligands – Schiff's bases, thiolates, polypyridyl and other polydentane ligands. Examples of some typical ligands of these classes are shown in Figure 1.14.
Schiff's bases are the classic example of the podand ligands. In 2002 Ford and coworkers synthesized several {RuNO}6 complexes using N,N'-bis(salicylidene)ethylenediamine (H2salen) as a ligand (Figure 1.14). Like the porphyrin ligand, compounds of this type contain four donor atoms, two nitrogen atoms in imine group and two phenolic oxygen atoms occupying four positions in the equatorial plane of the ruthenium polyhedron (Figure 1.15).
The study of the NO-release from these compounds under irradiation (Ȝ = 365 nm) showed a certain dependence on the nature of the solvent in which the process took place. Donor solvents such as acetonitrile have been shown to be able to stabilize the generated photoproduct, and the reverse process of the released NO-molecule coordination is relatively slow (k § 10-2-10-4 M-1s-1).77 If tetrahydrofuran (THF) or toluene (PhMe) is used instead of acetonitrile the process of photo-release is immediately followed by a very rapid recombination.80
N O NH N N N P aP y3H N NH O HN O N N Py3P H2
In this work it was also found the dependence of the photochemical activity of complexes on the ligand donor strength (Figure 1.15): with decreasing of ı-donating activity moving from Cl to H2O in the series ɋl -– ONO- – H2O quantum yield decreases as follows 0.13 - 0.067 - 0.005 respectively,.
Deep investigation of ruthenium-nitrosyl complexes with PaPy3H type ligands (Figure 1.14) were performed by Mascharak’s group. The principal feature of the structure of these ligands is the presence of the carboxamide nitrogen atom in its structure. The idea of using such a fragment was suggested by the structure of the enzyme nitrile hydratase.81 Two of the six donor atoms in the iron coordination polyhedron in active cite of this enzyme are carboxamide Nitrogen atoms, and its activity is regulated by the coordination of nitric oxide to the central iron atom.
Formation of the complex [(PaPy3)Ru(NO)]2+ with this ligand (Figure 1.16) occurs with deprotonation of the nitrogen atom of the carboxamide group. Thus, in the formed complex the negatively charged nitrogen atom acts as ı-donor in the trans-position to the nitrosyl ligand. Such an important nuance in the structure of the complex makes the compound stable in an aqueous medium at pH ranging from 5 to 9, despite the fact, that vast majority of ruthenium-nitrosyl coordination compounds undergo the transformation into nitrite form at pH>5:77
Ru–NO + H2O ї Ru–NO2 + 2H+
The absence of this transformation even in the Figure 1.14. Ligands
for the synthesis of ruthenium-nitrosil
complexes
Figure 1.15.
Cation of ruthenium nitrosyl complexes with
salen ligand N N OH HO H2salen N O NH HN O N H2bpb N S S SH HS py S4H2 N N O Ru O X NO X = Cl, ONO, H2O
presence of hydroxide anions indicates that a strong ı-donor ligand in the trans-position to nitric oxide in the {RuNO}6 complex protects the NO-group from the nucleophilic attack. Along with the high stability of [(PaPy3)Ru(NO)]2+ in solution, the compound also exhibits good photochemical properties. Nitric oxide is easily released from the compound (ij = 0.05), and the process of reverse recombination is not observed.77 These parameters (stability and NO-release
ability) make the compound interesting in terms of its direct use in biological research. Successful attempts to use [(PaPy3)Ru(NO)]2+ for the transport of NO to myoglobin and cytochrome oxidase are described by Mascharak group.82
Many works have recently been aimed at finding ways to sensitize ruthenium-nitrosyl complexes in the visible and infrared domains of light. For the purpose of the bathochromic shift of the transition dʌ(Ru) – ʌ*(NO), Mascharak group attempted to attach the ruthenium-nitrosyl complex to a molecule of a dye that absorbs light in the visible domain. Resorufin (Resf) and fluorescein derivatives (FlEt) were used as such molecules (Figure 1.17).
Figure 1.17. Dyes resorufin Resf (left) and fluorescein ethyl ester FlEt (right)
Using the ruthenium complex containing the dimethyl derivative H2bpb (Figure 1.14) as the basic platform, the researchers replaced the chlorine atom in the
trans-position to the nitrosyl group with the molecule of a dye (Dye) that was bound
to ruthenium atom through a covalent bond with hydroxyl group:83,84
Figure 1.16. Complex structure [(PaPy3)Ru(NO)]2+ HO O O COOEt FlE t O N OH O Resf N N N N Ru O N NO 2+
